上海地区幽门螺杆菌对克拉霉素耐药的相关基因检测  

Molecular mechanisms of clarithromycin resistance in Helicobacter pylori isolates in Shanghai

作　　者：黄声雷 郭玮 胡必杰[2] 周春妹 潘柏申 HUANG Shenglei;GUO Wei;HU Bijie;ZHOU Chunmei;PAN Boshen(Department of Laboratory Medicine,Zhongshan Hospital,Fudan University,Shanghai 200032,China)

机构地区：[1]复旦大学附属中山医院检验科,上海200032 [2]复旦大学附属中山医院感染科,上海200032

出　　处：《中国感染与化疗杂志》2019年第5期548-552,共5页Chinese Journal of Infection and Chemotherapy

摘　　要：目的通过荧光PCR熔解曲线方法直接检测临床样本中幽门螺杆菌(Hp)对克拉霉素耐药基因,了解上海地区Hp耐克拉霉素基因的分布特征,并为临床快速检测Hp对克拉霉素耐药性提供一种有效的方案。方法收集2017年9-12月复旦大学附属中山医院消化科就诊并接受胃镜检查的快速尿素酶试验Hp阳性患者的胃黏膜组织标本80份,使用荧光PCR熔解曲线法直接检测黏膜样本中Hp种特异性23SrRNA的区域并对扩增的核酸进行荧光标记,通过检测荧光变化绘制其熔解曲线,以确定Hp的23SrRNA突变情况;采用DNA测序技术直接检测标本中Hp克拉霉素23SrRNA耐药基因突变位点。并对两种方法的结果进行Kappa检验统计学比较。结果80份胃黏膜组织标本使用荧光PCR熔解曲线法共检测69份Hp阳性,11份Hp阴性,阳性率为86.2%。在69份Hp阳性标本中24份标本存在23SrRNA第2142或2143位点的变异,突变率34.8%。使用DNA测序法共检测出64份Hp阳性,阳性率为80.0%。其中23例患者的标本检测到23SrDNA耐药基因的突变,突变率35.9%。两种方法检测Hp耐药基因突变的阳性符合率、阴性符合率和总符合率分别为87.0%、95.1%和92.2%,Kappa系数为0.829,两者一致性较好。结论上海地区克拉霉素耐药Hp菌株基因型以23SrRNA基因的A2143G突变占主导。荧光PCR熔解曲线法可直接检测胃黏膜活检组织中Hp及其23SrRNA突变基因,该方法结果准确可靠。Objective The aim of this study was to investigate the distribution of clarithromycin-resistant genotypes of H.pylori in Shanghai and provide an effective solution for rapid detection of clarithromycin resistance in H.pylori.Methods We collected the samples of gastric mucosa from patients with positive rapid urease test in Department of Gastroenterology of Fudan University Zhongshan Hospital during the period from September to December 2017.Fluorescent PCR melting curve method was used to directly detect the region of H.pylori-specific 23S rRNA in samples and an anchor labeled with fluorescein which can hybridize several bases upstream from the former.After amplification,a melting step was performed leading to different melting points for the wild-type and mutants.23S rRNA in all strains were also investigated by sequencing of their PCR products.Results A total of 69(86.2%)samples were positive for H.pylori by fluorescent PCR melting curve method.Among the 69 specimens positive for H.pylori,24 specimens had 23S rRNA 2142 or 2143 mutations,with a mutation rate of 34.8%.Of 80 samples,64(80.0%)were positive for H.pylori by DNA sequencing.The mutation rate of 23S rDNA resistance gene was 35.9%in 23 of 64 H.pylori-positive samples.The main gene mutation associated with antibiotic resistance was 23S rRNA(A2143G),with mutation frequency of 100%(23/23).The mutations of H.pylori 23S rRNA in 64 positive samples were detected by both methods.The positive coincidence rate,negative coincidence rate and total coincidence rate were 87.0%,95.1%,and 92.2%respectively.The Kappa coefficient was 0.829.Conclusions Most clarithromycin-resistant H.pylori strains have A→G mutation on gene functional domain V2143 site of 23S rRNA.Fluorescent PCR melting curve can directly detect H.pylori status and its 23S rRNA mutation genes in gastric mucosa biopsy.This technique seems reliable and accurate.

关 键 词：幽门螺杆菌 克拉霉素耐药 荧光PCR熔解曲线法 DNA测序法 23SrRNA突变 

分 类 号：R378.2[医药卫生—病原生物学] R978.1[医药卫生—基础医学]